EPHA2 is a receptor tyrosine kinase that has a molecular weight of 130 kDa and has a single transmembrane domain (Molecular and Cellular Biology, 1990, vol. 10, p. 6316-6324). EPHA2 has a ligand-binding domain and two fibronectin type 3 domains present in the N-terminal extracellular region and a tyrosine kinase domain and a sterile-α-motif (SAM) domain present in the C-terminal intracellular region.
GPI-anchored plasma membrane proteins ephrin-A1 to ephrin-A5 are known as EPHA2 ligands (Annual Review of Neuroscience, 1998, vol. 21, p. 309-345). The ligand binding to EPHA2 activates the tyrosine kinase domain and phosphorylates tyrosine residues present in the EPHA2 intracellular region, resulting in signal transduction within the cell. It has also been reported that EPHA2 bound with the ligand is internalized into the cell through endocytosis and is eventually degraded by a proteasome (Molecular Cancer Research, 2002, vol. 1, p. 79-87).
High expression of EPHA2 has been reported clinically in many cancers, particularly, breast cancer, esophagus cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme (Cancer Research, 2001, vol. 61, p. 2301-2306; International Journal of Cancer, 2003, vol. 103, p. 657-663; The Prostate, 1999, vol. 41, p. 275-280; American Journal of Pathology, 2003, vol. 163, p. 2271-2276; Cancer Science, 2005, vol. 96, p. 42-47; Clinical Cancer Research, 2003, vol. 9, p. 613-618; Oncology Reports, 2004, vol. 11, p. 605-611; and Molecular Cancer Research, 2005, vol. 3, p. 541-551). It has also been reported that: for esophagus cancer, EPHA2 expression-positive patients tend to have a high frequency of regional lymph node metastasis, a large number of lymph node metastases, and a poor degree of tumor differentiation and a low five-year survival rate (International Journal of Cancer, 2003, vol. 103, p. 657-663); for non-small cell lung cancer, patients highly expressing EPHA2 tend to have a low disease-free survival rate and to have recurrence, particularly of brain metastasis (Clinical Cancer Research, 2003, vol. 9, p. 613-618); and for colon cancer, EPHA2 expression-positive patients tend to have liver metastasis, lymphatic vessel invasion, and lymph node metastasis, and many patients with high clinical stage are EPHA2 expression-positive patients (Oncology Reports, 2004, vol. 11, p. 605-611).
Moreover, it has been reported that by the introduction of EPHA2 genes into cells, non-cancer cells acquire cancer phenotypes such as anchorage-independent growth ability, tubular morphology-forming ability on the extracellular matrix, and in vivo tumor growth ability (Cancer Research, 2001, vol. 61, p. 2301-2306), and cancer cells have enhanced invasiveness through the extracellular matrix (Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102; and Oncogene, 2004, vol. 23, p. 1448-1456). In addition, it has been reported that: the invasiveness or anchorage-independent growth of cancer cells and in vivo tumor growth are inhibited by knockdown of EPHA2 expression using siRNA (Oncogene, 2004, vol. 23, p. 1448-1456; and Cancer Research, 2002, vol. 62, p. 2840-2847); and the invasiveness, anchorage-independent growth, and tubular morphology-forming ability of cancer cells are inhibited by activating EPHA2 using fusion proteins of its ligand ephrin-A1 and a human IgG Fc region and inducing EPHA2 degradation through endocytosis (Cancer Research, 2001, vol. 61, p. 2301-2306; Molecular Cancer Research, 2005, vol. 3, p. 541-551; and Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102).
On the other hand, EPHA2 has been reported to be expressed not only in cancer cells but also within tumors or in their surrounding blood vessels (Oncogene, 2000, vol. 19, p. 6043-6052). It has been reported that in mice, EPHA2 signals are involved in angiogenesis induced by ephrin-A1, and particularly, EPHA2 expressed in vascular endothelial cells is required for the tube formation or survival of the vascular endothelial cells (Journal of Cell Science, 2004, vol. 117, p. 2037-2049). It has also been reported that fusion proteins of an EPHA2 extracellular region and a human IgG Fc region inhibit angiogenesis in vivo and exhibit an antitumor effect (Oncogene, 2002, vol. 21, p. 7011-7026).
Monoclonal antibodies are useful not only as diagnostic drugs but also as therapeutic drugs. Monoclonal antibodies are actively used particularly in the field of cancer therapy, and monoclonal antibodies against receptor tyrosine kinases such as HER2 and EGFR or against CD20 extracellular regions are used in cancer therapy and exhibit excellent effects (The New England Journal of Medicine, 2007, vol. 357, p. 39-51; Oncogene, 2007, vol. 26, p. 3661-3678; and Oncogene, 2007, vol. 26, p. 3603-3613). The mechanisms of action of the monoclonal antibodies used in cancer therapy include apoptosis induction and inhibition of growth signals. In addition, their immunoresponse-mediated action such as ADCC or CDC is also considered to play a very important role. In actuality, it has been reported that anti-HER2 antibodies (trastuzumab) or anti-CD20 antibodies (rituximab) exhibit a much weaker antitumor effect in xenografts of nude mice deficient in FcγRs necessary for ADCC induction than in nude mice that are not deficient in FcγRs, when these antibodies are administered thereto (Nature Medicine, 2000, vol. 6, p. 443-446). It has also been reported that anti-CD20 antibodies (rituximab) exhibit a weaker antitumor effect in mice depleted of complement by the administration of cobra venom than in mice that are not depleted of complement, when the antibody is administered thereto (Blood, 2004, vol. 103, p. 2738-2743).
For EPHA2, it has been reported that agonistic anti-EPHA2 monoclonal antibodies having an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation, as for the ligands, inhibit the anchorage-independent growth of a breast cancer cell line and the tubular morphology formation thereof on the extracellular matrix (Cancer Research, 2002, vol. 62, p. 2840-2847). It has also been reported that agonistic anti-EPHA2 monoclonal antibodies which recognize an epitope on EPHA2 displayed on cancer cells rather than non-cancer cells and have an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation exhibit an antitumor effect in vivo (Cancer Research, 2003, vol. 63, p. 7907-7912; and the pamphlet of WO 03/094859). On the other hand, Kiewlich et al. have reported that their anti-EPHA2 monoclonal antibodies had an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation but did not exhibit an antitumor effect in vivo (Neoplasia, 2006, vol. 8, p. 18-30).
Moreover, the pamphlet of WO 2006/084226 discloses anti-EPHA2 monoclonal antibodies LUCA19, SG5, LUCA40, and SPL1 obtained by immunizing mice with cancer cells and discloses that, among these antibodies: LUCA19 and SG5 do not influence the phosphorylation of EPHA2 tyrosine residues; LUCA40 inhibits cancer cell growth in vitro; and LUCA19, SG5, and LUCA40 are internalized into cancer cells in the presence of anti-mouse antibody labeled with toxin (saporin). The document has also reported that LUCA40 and SPL1 exhibit an antitumor effect in vivo. However, the presence or absence of the agonistic activities of these antibodies remains to be clarified.
Despite these studies, an epitope for an anti-EPHA2 antibody that exhibits an antitumor effect in vivo is still unknown. No previous document has reported that a particular amino acid sequence in an EPHA2 extracellular region is useful as an epitope for a monoclonal antibody intended for cancer therapy.
Even antibodies against the same antigen differ in their properties depending on differences in epitopes or their sequences. Furthermore, due to this difference in their properties, the antibodies, when administered to humans, would clinically respond in different a manner with respect to drug effectiveness, the frequency of therapeutic response, side effects, the frequency of occurrence of drug resistance, etc.
Thus, a drug having clinically the best properties may also differ depending on the patient. In many cases, such properties are unknown until the drug is actually administered. Thus, it has been strongly required to develop a drug having a novel mechanism of action. It has also been strongly required to develop an antibody against EPHA2 having properties different from those of conventional antibodies.